Recent research into the proximal causes of Alzheimer disease has focussed on the processes involved in the formation of beta-amyloid. The principal proteinaceous component of amyloid, the 40-42 residue beta/A4 peptides, are generally thought to be cleaved from the beta-amyloid peptide precursor (beta-APP) by 2 proteases called beta-secretase and gamma-secretase. A third enzyme, alpha-secretase, cleaves beta-APP near the middle of the beta/A4 peptid sequence, and so may retard beta/A4 formation. Although many laboratories have sought to identify these enzymes, little has been learned of their properties because they function on the transmembrane beta-APP in the compartments of the secretory system. We discovered that Saccharomyces cerevisiae possesses alpha-secretases that resemble in their specificity and cellular localization the human alpha-secretase. We have generated yeast alpha-secretase mutants that transport beta-APP through the secretory system but cleave very little of it. These mutant strains are suitable hosts for the expression cloning of beta- and gamma-secretases. This proposal describes an approach to identify these enzymes. An invertase-negative yeast strain, deleted of its alpha-secretase genes, will be transformed with an episomal plasmid that expresses an APP-invertase fusion protein. This recombinant stain will serve as the host for a human brain cDNA library ligated into a centromeric expression plasmid. Presumptive secretase-expressing clones initially will be selected by their ability to grow on sucrose. Confirmation of their identities will be accompanied by ELISA assays that specifically identify alpha-, beta-, and gamma-secretases. The function of each putative secretase will be confirmed in mammalian cells by over- expression and by deletion of its mRNA. The study of the structure, function and regulation of these genes will become the subject for a subsequent proposal. The results of these investigations will contribute to the rational search for inhibitors and regulators of Abeta formation, which, according to the current consensus, would retard or eliminate the neuronal destruction of Alzheimer disease.